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Getting a Better Peek at Small Molecules inside Live Cells

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Scientists at Columbia University say they have made a major advance toward visualizing small biomolecules inside living biological systems with minimum disturbance. In a study published March 2nd in Nature Methods, Assistant A team led by Wei Min, Ph.D., professor of chemistry, reports that the development of a general method to image a broad spectrum of small biomolecules such as small molecular drugs and nucleic acids, amino acids, and lipids for determining where they are localized, and how they function inside cells.

The study (“Live-cell imaging of alkyne-tagged small biomolecules by stimulated Raman scattering”) is published in Nature Methods.

When studying biological functions of a molecule in complex cells, researchers typically label the molecules of interest with fluorophores. Using a fluorescence microscope, the fluorophore-tagged molecules can be located and tracked. The invention of green fluorescent protein (GFP), in 1994, compatible with imaging inside live cells and animals, has since made fluorescence microscopy even more popular.

However, when it comes to small biomolecules, fluorophore tagging is problematic, because the fluorophores are almost always larger or comparable in size to the small molecules of interest. As a result, they often disturb the normal functions of these small molecules with crucial biological roles.

To address this problem, Dr. Min and his group departed from the conventional paradigm of fluorescence imaging of fluorophores, and pursued a novel combination of physics and chemistry. Specifically, they coupled an emerging laser-based technique called stimulated Raman scattering (SRS) microscopy with an alkyne tag, a chemical bond that, when it stretches, produces a Raman scattering signal at a unique frequency.

This new technique, labeling the small molecules with this tiny alkyne tag, avoids perturbation that occurs with large fluorescent tags, while obtaining high detection specificity and sensitivity by SRS imaging, according to Dr. Min. By tuning the laser colors to the alkyne frequency and quickly scanning the focused laser beam across the sample point by point, SRS microscopy can pick up the unique stretching motion of the C=C bond carried by the small molecules and produce a three-dimensional map of the molecules inside living cells and animals.

“Sensitive and specific visualization of small biomolecules in living systems is highly challenging,” wrote the investigators. “We report stimulated Raman-scattering imaging of alkyne tags as a general strategy for studying a broad spectrum of small biomolecules in live cells and animals. We demonstrate this technique by tracking alkyne-bearing drugs in mouse tissues and visualizing de novo synthesis of DNA, RNA, proteins, phospholipids, and triglycerides through metabolic incorporation of alkyne-tagged small precursors.”

“The major advantages of our technique lie in the superb sensitivity, specificity, and biocompatibility with dynamics of live cells and animals for small molecule imaging,” says the lead author Lu Wei, a Ph.D. candidate in chemistry.

Next, Dr. Min’s team will apply this new method to biomedical questions, such as detecting tumor cells and probing drug pharmacokinetics in animal models. They are also creating other alkyne-labeled biologically active molecules for more versatile imaging applications.

“Our new technique will open up numerous otherwise difficult studies on small biomolecules in live cells and animals,” explained Dr. Min. “In addition to basic research, our technique could also contribute greatly to translational applications. I believe SRS imaging of alkyne tags could do for small biomolecules what fluorescence imaging of fluorophores such as GFP has done for larger species.”

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